Study of phylogenetic relationships between twelve species of cestoda parasites by using Internal transcribed spacer2 (ITS2) and 5.8S ribosomal genes
A Valappil, M Pillai, Y Mundaganur, D Mundaganur, S Angadi
5.8s rdna, cestodes, clutalw, motifs and two-dimensional histogram, ribosomal internal transcribed spacer 2 its2
A Valappil, M Pillai, Y Mundaganur, D Mundaganur, S Angadi. Study of phylogenetic relationships between twelve species of cestoda parasites by using Internal transcribed spacer2 (ITS2) and 5.8S ribosomal genes. The Internet Journal of Parasitic Diseases. 2008 Volume 4 Number 1.
Cestodes are parasitic flatworms causing disease of diverse nature in animals belongs to all the phyla including man. The phylogenetic status of the cestodes remains controversial for long time. In this contest a comparative study of common core secondary structure in the ribosomal internal transcribed spacer2 (ITS2) and 5.8SrDNA gene of 12 selected cestodes species from different hosts was carried out. Multiple sequence alignment and secondary structure analysis of ITS2 and 5.8S rDNA was performed to elucidate the phylogenetic relationships. This study reveals a phylogenetic relationship among the different species of cestodes from different hosts; with some of them support by compensatory changes, suggesting the significant role by ITS2 an RNA domain during ribosomal biogenesis. The sudy also shows that among the selected species
Internal Transcribed spacers are widely used to resolve the phylogenetic relationship for closely related taxa due to their relatively rapid evolution rate (Baldwin, 1992; Schlotterer et al, 1994; Mai and Coleman, 1997; Weekers et al, 2001; Oliver et al, 2002, Ashokan and Pillai, 2008). Internal transcribed spacer refers to a piece of non-functional RNA situated between structural ribosomal RNA on a common precursor transcript. Many parasitologists used internal transcribed spacers and ribosomal DNA as tool to resolve the controversial phylogenetic dispute of cestod parasites (Skerikova et al, 2007; Luo et al, 2003; Literak et al, 2006; Kralova et al, 2001;Magnish et al, 2002; Wickstron et al, 2005; Gao et al, 2007; Logan et al, 2004; Haukisalmi et al, 2008 ). Skerikova et al, 2007 used 18SrDNA to investigate phylogeny of European species of tapeworm genus Proteocephalus. The study of Luao et al (2003) in the genus Digramma and Ligula (Pseudophyllidea) using the entire internal transcribed spacer of the ribosomal DNA showed low level of nucleotide variation between the two genera may imply that cestodes in the genus Digramma is synonym of Ligula. The phylogenetic study of 20 samples of pseudophyllidea cestodes of family Diphyllobothridea from different hosts and geographical region using ITS2 rRNA shows controversy in the previous phylogeny of these groups (Logan et al, 2004). The phylogenetic study of
The transcripts folding structure of the ITS2 provide some signals that guide the ribosomal coding region when they are processed into small, 5.8S and large ribosomal RNA (Van der Sande
The over all literature cited shows that ITS2 are widely used to resolve the phylogeny of cestodes and many other invertebrates. Utility of ITS2 in phylogenetic study is limited with regards to particular species of cestodes belongs to limited families. Thus the present investigation was concentrated to reveal the phylogeny of cestodes from different families using ITS2 and 5.8S rDNA genes.
Materials and Methods
ITS2 and 5.8S rDNA sequences of 12 cestodes from eucestoda belongs to different orders (Pseudocephalidea, Cyclocephalidea, Proteocephalidea, Anaplocephalidea and Cyclocephalidea) that are deposited in GenBank were investigated. The accession numbers of ITS2 and 5.8S gene are:-
Multiple sequence alignment were performed by using ClustalW with gap open penalty 15 and gap extension penalty 07.This program align nucleotides using a progress alignment algorithm(Feng and Doolittle, 1987).
The motifs in the sequence of ITS2 were finding using the SSRT (Simple sequence repeater identification tool). This tool finds all perfect simple sequence repeats (SSRs) in a given sequence (Temnykh et al 2001).
Secondary structure prediction
The RNA secondary structure for ITS2 and 5.8S were predicted by using RNADRAW online software (Christoffersen
The phylogenetic service of ClustalW was used for phylogenetic tree construction Unweighted Pair Group Method using Arithmetic average (UPGMA) (Michener and Sokal, 1957; Sneath and Sokal, 1973). Clustering algorithm was used, for interpreting phylogenies bootstrap values are used.
Sequence analysis of ITS2 and 5.8S regions
The length of ITS2 of 12 selected cestodes ranged in size 444bp and 677bp. Twelve dispersed but unambiguously conserved sequence segments encompassing about a third of the ITS2 length have been identified (Fig1). They were interspersed with variable regions and gaps where size variations accumulate. The characteristics of sequences for each isolates are shown in the table 1. The length variations were observed with maximum length being 677bp in
Secondary structure in ITS2 and 5.8S regions
Secondary structures of ITS2 and 5.8S regions were given in Table 2. The secondary structure of the cestodes from different hosts belonging to different order was classified into four groups based on the analysis of conserved stem and loops (Fig 2). Class I includes
Tapeworms are a group of parasitic worms that live in the intestinal tracts of some animals including man. Several different species of tapeworms can infect human. Tapeworm infection pose serious public problem in many less developed countries due to poor sanitation conditions (Garcia et al, 2003). The tapeworms are biologically known as
cestodes. The phylogeny of cestodes is one of the most controversial one in front of systematists. Monophyly of euestoda was firmly corroborated by the study of Hoberg et al (1997). The molecular study reveals that the morphological charcter based classifications of the cestodes are controversial (Lao et al, 2003). The low level of nucleotide relation between the two genera may imply that cestodes in the genus
In the present investigation, the ITS2 and 5.8S rDNA sequence reflected the trend observed in phylogeny. The more distantly related the less was the convergence at the ITS2 level (Figure-1 and 2). However accumulated substitutions in ITS2 leading to length variation also had a profound effect on the conserved ness among the structure. The length of ITS2 is more or less similar in all the isolates except
The selected 12 cestodes species from different hosts present world wide and they cause diseases of various extensions in their hosts. This study reveals although sequence variation was found in ITS2 sequence this did not correlate well with morphology and host suggesting that all the sequences studied belong with a single polymorphic species. All the selected species shows sequence alignment considerably and reflected the similarity in energy level and secondary structure. While the difference between the species is to rank them in different orders is substantiated through motif structure and different loops in the secondary structure of both ITS2 and 5.8S rRNA